A model for the regulation of Tim and related GEFs by intramolecular and intermolecular interactions is depicted in . In the basal state (left), the exchange potential of the GEF is autoinhibited by two sets of intramolecular interactions: (i) the autoinhibitory helix packing into a conserved pocket on the DH domain and (ii) the SH3 domain binding to the N-terminal polyproline region. These two sets of interactions are mutually cooperative and serve to tightly suppress access of Rho GTPases to the surface of the DH domain needed to catalyze guanine nucleotide exchange. Binding of another protein to the SH3 domain, with an affinity higher than that of the intramolecular interaction, would remove the SH3 domain from that polyproline region (center). This binding interaction could also localize the GEF to the appropriate subcellular compartment for nucleotide exchange. Interactions between another as yet unidentified protein and the polyproline region of the GEF would similarly serve to modulate GEF activity and localization.
FIGURE 7 Model for the regulation of Tim and its paralogs by intramolecular and intermolecular interactions. In the basal state (left), the exchange potential of Tim is autoinhibited by two sets of intramolecular interactions. First, the autoinhibitory helix (red) (more ...)
With interactions between the SH3 domain and the polyproline region disrupted, interaction of the autoinhibitory helix with the DH domain would be destabilized, increasing the frequency that the autoinhibitory helix is solvent-exposed. Src could then phosphorylate the tyrosine residues in the autoinhibitory helix as previously described (11
), both preventing a rebinding event and fully activating the exchange potential of this Dbl-family GEF ().
Activation of Rho GTPases downstream of transmembrane receptors such as Ephs occurs through either inactivation of GAPs or activation of GEFs. While EphB receptors are able to interact with Intersectin (31
), Kalirin (2
), and Vav2 (32
), the best-characterized interaction between an EphA receptor and a Dbl protein is that of EphA4 and ephexin. Ephexin is tyrosine phosphorylated in an N-terminal motif with a significant degree of sequence identity to the autoinhibitory helix described here for Ngef and related Dbl-family members. Phosphorylation of ephexin in this motif is reported to change the specificity of this GEF for cognate GTPases. Specifically, when ephexin was not tyrosine phosphorylated, it activated RhoA, Rac1, and Cdc42, but when ephexin was tyrosine phosphorylated, ephexin activated RhoA exclusively (15
). These results, based largely upon examination of the cellular morphology of fibroblasts transiently transfected with mutants of ephexin and EphA4, are in contrast to those shown here.
We have used in vitro guanine nucleotide exchange assays and cell-based GTPase activity assays to show mechanistically that phosphorylation or phosphomimetic mutation of Ngef leads to an activation of the exchange potential of this GEF toward all three GTPases that were studied: RhoA, Rac1, and Cdc42. This discrepancy between human Ngef and it mouse ortholog, ephexin, could be explained in several different ways. First, phosphorylated Ngef may be differentially localized in the growth cone to activate Rac1 and Cdc42 in restricted microdomains. High-resolution microscopy studies examining the intracellular localization of phosphorylated Ngef and the activated cognate GTPases need to be carried out to examine this idea. Second, ephrin A binding to EphA4 could independently activate an as yet unidentified GAP for Rac1 and Cdc42 such that the net result of this signaling pathway is a preferential activation of RhoA. Finally, activation of Rac1 and Cdc42 by Ngef could play a role in growth cone collapse. Rac1 activity has been shown to be required for growth cone collapse since it promotes internalization of the Eph-ephrin complex from the plasma membrane (32
). Additionally, active Rac1 and Cdc42 are critical for axon retraction, branching, and defasiculation following growth cone collapse (34
). The role of Ngef in these processes has yet to be determined.
Further work is necessary to identify potential intermolecular binding partners for the SH3 domains and polyproline regions of the members of this subfamily. One potential binding partner for the polyproline region of Tim is the SH3 domain of Src itself. In this scenario, the interaction between Src and the polyproline region of Tim would relieve the autoinhibitory interactions between the SH3 domain of Src and its intramolecular ligand in the SH2 kinase linker, thus allowing the kinase domain to phosphorylate and activate Tim. This positive feedback loop would allow for rapid, specific activation of Tim.
Phenotypic diversity and complexity in biological systems often arise from new combinations of proteins and independently folding protein domains working together in networks, and not from de novo generation of new protein functions (35
). In fact, multiple-sequence analysis of Dbl-family proteins from diverse animal species revealed that GEFs are composed of an N-terminal SH3 domain followed by a DH/ PH cassette, such as Asef, and GEFs are composed of a DH/PH cassette followed by an SH3 domain, such as Tim, evolved from a common ancestor composed of only a DH/PH cassette through independent insertion of the SH3 domain (36
). Since the SH3 domains of both Tim and Asef function to regulate the exchange potential of these GEFs by autoinhibition, it is likely that the evolutionary pressure for the insertion of the SH3 domain was to achieve finer control of the exchange activity of these Dbl-family proteins.
The stabilization of the autoinhibitory motif on the DH domain of Tim by intramolecular interactions between the SH3 domain and polyproline regions, like the autoinhibitory motif itself, is reminiscent of the mechanism of autoinhibition of the Vav isozymes. For example, recent structural characterization of full-length Vav3 by electron microscopy showed that the CH domain of this protein stabilizes the acidic region, including the autoinhibitory helix, through intramolecular interactions with the zinc finger domain (37
). For both the Tim and Vav subfamilies, then, multiple domains are functioning together to stabilize the inactive state of the catalytic DH domain. While the first set of interactions involving the autoinhibitory helix is conserved between the Tim and Vav subfamilies (11
), the second set of interactions is divergent in both primary and tertiary structure. If autoinhibition by interaction of the autoinhibitory helix with the DH domain is a conserved mechanism of regulation among Dbl-family GEFs, it seems likely that the second, cooperative set of stabilizing interactions dictates the activating inputs and fine-tunes the dynamics of the guanine nucleotide exchange process. In this sense, the two sets of autoinhibitory interactions with Tim and Vav approximate a logical AND gate (38
). Ultimately, full activation of these Dbl-family proteins cannot occur until both sets of autoinhibitory interactions are removed, thus allowing a large degree of spatiotemporal control over the activation of GTPases by Dbl-family proteins. We anticipate similar coordinated regulation to be found in other members of the Dbl family.
In conclusion, we have demonstrated that Ngef is activated toward its full repertoire of cognate GTPases, namely RhoA, Rac1, and Cdc42, by removal, substitution, or Src-dependent tyrosine phosphorylation of a small, conserved sequence N-terminal to its DH domain. Similarly, EphA4-dependent phosphorylation of Ngef promotes the activation of multiple Rho GTPases. Ngef exchange activity is inhibited by a peptide derived from the autoinhibitory sequence of the related protein, Tim, indicating that these two proteins are regulated in a nearly identical manner. A third member of this subfamily, Wgef, is regulated similarly. Tim, Ngef, and Wgef cluster with Sgef, Vsm-RhoGEF, and neuroblastoma on the basis of common domain architecture and sequence similarity. Given that each of these Dbl-family members shares highly conserved autoinhibitory motifs, it is highly likely that all members of this clade are regulated by steric occlusion of their DH domains by short helical motifs and that this autoinhibition is relieved physiologically by phosphorylation of these motifs. Finally, we have identified a second mechanism of autoinhibition that is likely to be conserved among the members of this clade, which is mediated by interactions between the C-terminal SH3 domain and N-terminal polyproline region and may function to increase the degree of spatiotemporal control over the activation of the Tim-related proteins.